Many aspects of biological research rely upon the ability to perform extremely large numbers of chemical and biochemical assays. Increasing the throughput of screening assays has allowed researchers to adopt a more generalized approach to the overall screening process, as opposed to a more rational, predefined process. For example, in the pharmaceutical discovery process, large libraries of different compounds are generally screened against defined target systems to determine whether any of those compounds have a desired effect on that system. Once a compound is identified to have the desired effect, it is then subjected to more rigorous analysis.
Many high-throughput screening assay systems rely upon entirely in vitro models of biological systems. This is due, at least in part, to the ability to accurately control substantially all of the parameters of the model system that is being assayed to permit correlation from assay to assay, such as the quantity and purity of reagents, the environmental conditions of the assay, the operator performing the assay, and the like. Specifically, variation of any of these parameters can produce widely varying results in the performance of a given assay.
In many cases, these in vitro systems have proven to be effective models of the biochemical system of interest, and have led to the identification of promising pharmaceutical candidate compounds. However, in many instances it is desirable to use a model system that is a closer representation of what actually occurs in more complex systems, e.g., in vivo. Cell-based systems offer a closer model to these relevant biological systems, and have generally been widely adopted as screening assays. While cell-based assays are generally preferred in screening applications, these assays have proven somewhat difficult to adapt to conventional notions of high-throughput and even ultra high-throughput screening assay systems.
As the multiplicity of cell-based assays increases, it becomes extremely advantageous to miniaturize the assay geometry. In the first instance, this miniaturization increases the efficiency of the assay by optimizing space utilization, reducing assay volumes, and consequently reduces reagent consumption and assay costs. For example, cells themselves, being a consumed reagent in such assays, are an expensive and perishable component of these assays, and quickly become a limiting influence on the application of these assays to high-throughput systems. Again, by miniaturizing assay geometries, the amount of this consumable reagent is reduced.
In addition to the economies of miniaturization, described above, a number of assay parameters become more and more critical as assay volumes are decreased. First, cells require a nutrient medium having a controlled pH in sufficient quantity to sustain their continued viability. Second, the cells need to be protected from desiccation, which is a particular problem in very small fluid volumes. Third, otherwise simple manipulations, such as reagent addition, rapid mixing and sampling become very difficult when dealing with extremely small fluid volumes. Further, continuous and accurate kinetic reading of assay results, e.g., monitoring of signals, during and after reagent or sample addition is a necessary element of many cell-based assays. Often these assay results come in the form of very small changes in signal levels from the cells, e.g., intracellular or membrane associated fluorescent signals. These small changes become increasingly difficult to detect as assay volumes are decreased and signal to noise ratios decrease.
Accordingly, it would generally be desirable to provide methods, devices and systems for performing cell based assays that are readily adaptable to high throughput screening applications, are readily automated, are easily repeated, and require less reagents and/or other assay components. The present invention meets these and a variety of other needs.
In a first aspect, the present invention provides methods of determining a function of cells, which comprises a suspension of cells flowing along a first fluid channel. The cells have a first detectable property associated therewith, and wherein the cells produce a second detectable property upon activation of the function of the cells, the first and second detectable properties being distinguishable from each other. The levels of the first and second detectable properties are measured. The level of second detectable property is compared to the level of first detectable property to determine the relative function of the cells.
The present invention also provides an apparatus for measuring a function of cells, comprising a body structure having a first fluid channel disposed therein. The first fluid channel is in fluid communication with a first source of a suspension of cells and the cells have a first detectable property associated therewith. The cells produce a second detectable property upon activation of the function of the cells, the first and second detectable properties being distinguishable from each other. The apparatus also optionally includes a material transport system for flowing the suspension of cells along the first channel and a detector for detecting and distinguishing the first detectable property from the second detectable property associated with cells within the first channel.
The present invention also provides methods of measuring a binding function of a cell, comprising a channel disposed in a first body structure. The channel comprises a first binding region and a non-binding region, the first binding region having a binding moiety immobilized on an interior surface of the first channel therein. A suspension of cells flows along a first channel, the cells comprising on their surfaces, a moiety specifically bound by the binding moiety. A relative velocity of cells flowing through the binding region is determined, relative to a velocity of cells flowing through a non-binding region. A decrease in the relative velocity is indicative of first binding in the binding region.
The present invention also provides an apparatus for measuring a binding function of a cell, using a body structure comprising a first channel disposed therein. The channel includes a binding region, a non-binding region, a binding moiety immobilized on an interior surface of the first channel in the binding region but not the non-binding region, a source of a suspension of cells in fluid communication with the first channel, a means for flowing the suspension of cells along the first channel, and a detection system for determining a relative velocity of cells flowing through the binding region compared to a velocity of cells flowing through the non-binding region.